Luminescent/paramagnetic probes for detecting order in biological assemblies: transformation of luminescent probes into pi-radicals by photochemical reduction.

The spectroscopic methods of fluorescence polarization and electron paramagnetic resonance (EPR) are used to study order and orientation of extrinsically labeled protein elements of ordered biological systems. These methods generate complementary information about the order of the system, but a consistent quantitative interpretation of the related data is complicated because the signals arise from different donors. We introduce a new method that allows us to detect both signals from the same donor. Unsubstituted xanthene dyes (eosin, erythrosin, and fluorescein) were irradiated by laser light at their absorption maximum in the presence of different reducing agents. Due to photochemical reduction, the quinoidal structure of the xanthene ring is transformed into a semiquinone, and a pi-radical is formed having a characteristic EPR signal of an unpaired electron spin with proton hyperfine interactions. A strong EPR signal is observed from the dye in solution or when specifically attached to a protein following irradiation in the presence of dithiothreitol or cysteine. We applied this technique to the study of skeletal muscle fibers. The fluorescent dye (iodoacetamido)fluorescein was covalently attached to the reactive thiol of the myosin molecule in muscle fibers. Fluorescence polarization and EPR spectroscopy were performed on the labeled fibers in rigor. Both signals indicate a highly ordered system characteristic of cross-bridges bound to actin. Our use of the same signal donor for fluorescence and EPR studies of probe order is a promising new technique for the study of order in protein elements of biological assemblies.